Description

Nori Human OCLN ELISA Kit Summary

Alternative names for OCLN: Occludin

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Occludin (OCLN) is a protein that in humans is encoded by the OCLN gene.^[1] Occludin is a 65-kDa integral plasma-membrane protein located at the tight junctions, described for the first time in 1993 by Shoichiro Tsukita.^[2] Together with the Claudin group of proteins, it is the main component of the tight junctions. Occludin is important in tight junction stability and barrier function. Studies in which mice were deprived of occludin expression showed morphological stability in several epithelial tissues but also found chronic inflammation and hyperplasia in the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in straited duct cells of salivary gland, and thinning of the compact bone. The phenotypical response of these mice to the lack of occludin suggest that the function of occludin is more complex than thought and requires more work.^[3] Occludin plays a critical role in maintaining the barrier properties of a tight junction. Thus, mutation or absence of occludin increases epithelial leakiness which is an important barrier in preventing metastasis of cancer. Loss of occludin or abnormal expression of occludin has been shown to cause increased invasion, reduced adhesion and significantly reduced tight junction function in breast cancer tissues. Furthermore, patients with metastatic disease displayed significantly lower levels of occludin suggesting that the loss of occludin and thereby loss of tight junction integrity is important in metastatic development of breast cancer.^[4] Occludin also plays an important role in the apoptosis. The C-terminus of occludin is important in receiving and transmitting cell survival signals. In standard cells, loss or disruption of occludin and other tight junction proteins leads to initiation of apoptosis through extrinsic pathways.^[5] Studies involving high levels of expression of occludin in cancer cells have shown that occludin mitigates several important cancer proliferation properties. The presence of occludin decreased cellular invasiveness and motility, enhanced cellular sensitivity to apoptogenic factors and lowered tumorigenesis and metastasis of the cancer cells. Specifically, occludin has a strong inhibitory effect on Raf1-induced tumorigenesis. Still, the exact mechanism of how occludin prevents the progression of cancer is not known but it has been shown that cancer progression is linked to the loss of occludin or the silencing of the OCLN gene.^[6]

References

1. Ando-Akatsuka Y, et al. (1996). J Cell Biol. 133 (1): 43–47.
2. Furuse M, et al. (1993). J. Cell Biol. 123 (6 Pt 2): 1777–1788.
3. Saitou M, et al. (2000). Molecular Biology of the Cell. 11 (12): 4131–42.
4. Martin TA, et al. (2010). International Journal of Molecular Medicine. 26 (5): 723–34.
5. Beeman N, et al. (2012). Cell Death & Disease. 3 (2): e273.
6. Osanai M, et al. (2006). Cancer Research. 66 (18): 9125–33.

Product Citations